The present invention relates to an improved assay for determining the effects of activated protein C ("APC") in test samples to ensure rapid and accurate evaluations. A test sample includes any material that is expected to possess the capability to function with activated protein C. These materials are typically plasma or plasma fractions.
The present assay is based on measuring the conversion of factor X to activated factor X ("factor Xa") by activated factor VIII ("factor VIIIa") present within the test solution. APC inhibits this conversion.
Protein C is a vitamin K-dependent glycoprotein that is synthesized in the liver and circulates in plasma as an inactive zymogen at a concentration of about 4 .mu.g/ml. This protein is converted into an active serine protease (APC) by the thrombin-thrombomodulin complex on the surface of the vessel wall (endothelium). Protein C also may be activated by non-physiologic enzymes, such as the snake-venom factor (Protac C.RTM., Pentapharm, Switzerland)
APC has fibrinolytic properties imparted by its ability to inactivate plasminogen activator inhibitor. APC also has an anticoagulant effect because it can proteolytically degrade factor Va and factor VIIIa. Factor Va is a cofactor for the factor Xa-induced prothrombin activation to thrombin. Factor VIIIa is the cofactor of factor X conversion to factor Xa. As stated above, factor Xa along with factor Va are involved in the conversion of prothrombin to thrombin. Accordingly, the activation of protein C in vivo to form APC constitutes a negative feedback reaction in the generation of thrombin via the degradation of factors Va and VIIIa.
The sensitivity of an individual to the normal activity of activated protein C is defined as "APC sensitivity." APC sensitivities may be evaluated with test samples, such as plasma samples or fractions, obtained from a given individual. Plasma samples or fractions from individuals that are highly sensitive to APC show marked anticoagulation activity when APC is administered. Plasma samples from individuals with decreased APC sensitivity exhibit minimal anticoagulation activity.
A co-factor, protein S, is needed to develop the optimum APC activity. Protein S is a non-enzymatic cofactor for the anticoagulating and pro-fibrinolytic properties of APC. In the plasma, protein S is present in various forms. These forms include a free, active form and an inactive form, which is non-covalently complexed with the C4b-binding protein. In a protein S depleted plasma, APC cannot perform its normal activity. APC will recover its normal activity after addition of protein S.
APC increases the plasma clotting time in a dose-dependent manner. The pathophysiological role of APC is demonstrated in individuals suffering from thrombophilia caused by a congenital protein S or protein C deficiency. Congenital protein S deficiency is autosomal-dominantly inherited, and is characterized by the occurrence of venous and arterial thromboembolisms in early youth. Other adverse effects caused by APC abnormalities include microvascular coagulation, reperfusion injury and septic shock. See Esmon, TCM 2: 214-19 (1992).
A variety of causes may be responsible for decreased APC sensitivity. For example, Dahlback et al. have discovered a syndrome characterized by poor anticoagulant response to APC. See Proc. Nat'l Acad. Sci. USA 90: 1004-08 (1993). The blood of patients with this syndrome were found to have normal levels of antithrombin III, protein C, protein S, plasminogen, fibrinogen and other coagulation factors. Dahlback et al. postulated that a previously unrecognized cofactor of APC is deficient in the plasma of patients with this syndrome. This syndrome is thought to be inherited. Reliable assays for measuring APC are needed due to the existence of such syndromes.
Tests have been previously developed to assess the blood coagulation disorders of an individual. Methods are described in WO 93/10261. This assay is based on combining an activator of the blood coagulation system with APC and auxiliary agents, such as phospholipids and calcium ions. In this method, the anticoagulant response to human APC is evaluated with factor IXa and factor Xa based coagulation assays. This test is stated to permit detection of disorders not caused by protein S deficiencies of APC resistant factor Va and VIIIa.
The assay of WO 93/10261 involves the entire coagulation cascade, and employs more than one incubation step. This assay requires high concentrations of plasma because it involves the beginning of the coagulation cascade (kallikrein). These concentrations are more than 10% v/v plasma sample, usually as high as 20-35% v/v. The high concentrations of plasma in the reaction mixture permits the factors within the sample to interfere with the assay and thereby decrease its sensitivity and specificity.
Factor VIII activity assays have some applicability in the evaluation of APC sensitivity. One such assay is described in U.S. Ser. No. 07/819,456. The reagent used in this assay contains factor IXa.beta., factor X, calcium ions, thrombin, phospholipids and, if desired, factor XIa and factor XIIa. This assay is based on activation of factor X as a function of factor VIII contained in the sample. Factor Xa is then quantitatively determined.
When this assay is used to evaluate proteins that react with factor VIII, such as APC and its cofactor protein S, an excess amount of factor VIII is added to the sample and reacted. The reaction decreases factor VIII activity, and the remaining amount of factor VIII is then determined using a factor VIII chromogenic reagent. In the context of determining APC sensitivity, however, it is desirable to maintain the ratio of substances which influence this activity, such as factor VIII, similar to the levels found in plasma. Additionally, in the context of APC sensitivity this method requires more than one incubation step.